Mutant mono-oxygenase cytochrome P450cam

ABSTRACT

A mutant of the mono-oxygenase cytochrome P450cam in which the cysteine residue at position 334 is removed.

The present invention relates to a mutant of the mono-oxygenase cytochrome P-450cam.

BACKGROUND OF THE INVENTION

Mono-oxygenases catalyse the selective oxidation of activated and unactivated carbon-hydrogen bonds using oxygen¹, and are therefore of great interest for potential use in organic synthesis. However, progress in this area has been hampered by the difficulty in isolating sufficient quantities of the mono-oxygenase enzyme and/or the associated electron-transfer proteins. Despite the availability of amino acid sequences of more than 150 different cytochrome P-450 mono-oxygenases, to date structural date of only three are available².3.4, and few have been successfully over-expressed in bacterial systems⁵.

One cytochrome P-450 mono-oxygenase, which is soluble and can be expressed in sufficient quantities, is the highly specific P-450cam from P. putida which catalyses the regio- and stereo-selective hydroxylation of camphor to 5-exo-hydroxycamphor⁶. The high resolution crystal structure of P-450cam has been determined², and since the mechanism of action of this bacterial enzyme is believed to be very similar to that of its mammalian counterparts, it has been used as a framework on which structural models of mammalian enzymes are based.

The nucleotide sequence and corresponding amino acid sequence of P-450cam have been described⁵.7. The location of an active site of the enzyme is known and structure-function relationships have been investigated⁸.9. Mutants of P-450cam have been described at the 101 and 185 and 247 and 295 positions⁹.10.11. and at the 87 position¹². A mutant in which tyrosine 96 (Y96) has been changed to phenylalanine 96 (the Y96F mutant) has been described¹¹.13.14.15.. But in all cases the papers report effects of the mutations on the oxidation reactions of molecules which had previously been shown to be substrates for the wild-type enzyme. There is no teaching of how mutations might be used to provide biocatalysts for oxidation of different, novel substrates.

SUMMARY OF THE INVENTION

In an attempt to develop new biocatalysts, we have initiated a project which aims to redesign P-450cam, such that it is able more effectively to carry out specific oxidations of organic molecules whether or not these are substrates for the wild-type protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that the three dimensional structure of P-450cam shows the active site to provide close van der Waals contact with the hydrophobic groups of camphor.

FIG. 2A and FIG. 2B are gas chromatographs in accord with the present invention.

The three dimensional structure of P-450cam shows the active site to provide close van der Waals contacts with the hydrophobic groups of camphor as shown in FIG. 1. Of particular significance are the contacts between camphor and the side chains of leucine 244, valine 247 and valine 295. Three aromatic residues (Y96, F87 and F98) are grouped together and line the substrate binding pocket, with a hydrogen bond between tyrosine 96 and the camphor carbonyl oxygen maintaining the substrate in the correct orientation to ensure the regio- and stereo-specificity of the reaction.

Lipscomb and co-workers¹⁶ demonstrated in 1978 that wild-type P-450cam showed a propensity to dimerise, but they also reported that the catalytic activity of the monomer and dimer towards camphor oxidation were indistinguishable. Since the dimerisation reaction could be reversed by thiol reducing agents, they concluded that it occurred by intermolecular cysteine disulphide (S--S) bond formation. They were unable to determine whether dimerisation involved more than one cysteine per P-450cam molecule. Nor were they able to identify the key cysteine residue(s) involved in this reaction because neither the amino acid sequence nor crystal structure of P-450cam were known at the time.

We used molecular modelling to investigate the likely effects of points mutations to the three aromatic residues (Y96, F87, F98) in the active site pocket. We noted that replacement of any of these aromatic residues with a smaller, hydrophobic non-aromatic side-chain could provide an "aromatic pocket" which could be used to bind more hydrophobic substrates. The program GRID¹⁷ was used to calculate an energy of interaction between an aromatic probe and possible mutants of cytochrome P-450cam where these residues were changed to alanine (F87A, Y96A and F98A). The results were then examined graphically using the molecular modelling package Quanta¹⁸.

The mutant F98A appeared to have the strongest binding interaction within the active site cavity accessible to the aromatic probe, with that of Y96A being slightly smaller, and that of F87A being substantially less. It was decided in the first instance to mutate tyrosine 96 to alanine as it is more central to the binding pocket, whereas phenylalanine 98 is in a groove to one side. Also, removal of tyrosine 96 should decrease the specificity of the enzyme towards camphor due to the loss of hydrogen bonding to the substrate.

According to one aspect of the present invention a mutant of the mono-oxygenase cytochrome p-450cam is provided in which the cysteine residue at position 334 is removed.

Preferably the removal is by the substitution of another amino acid except cysteine for the cysteine residue.

Alternatively the removal is by the deletion of the entire cysteine 344 residue from the enzyme.

Suitably the tyrosine residue at position 96 in the mutant is replaced by the residue of any amino acid except tyrosine.

Conveniently the amino acid is selected from any one of the following: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine and valine except that in the case of the cysteine residue at position 334, the amino acid is not cysteine and in the case of the tyrosine residue at position 96 the amino acid is not tyrosine.

Preferably the amino acid residue at one or more of the positions 87, 98, 101, 185, 193, 244, 247, 295, 297, 395 and 396 is replaced by another amino acid residue.

We examined the structure of P-450cam generated from the published crystallographic atomic co-ordinates using the modelling programme Quanta. We determined that there are five cysteines near the surface of P-450cam (cysteines 58, 85, 136, 148, 334) which might participate in intermolecular disulphide bond formation leading to protein dimerisation. We carried out sit-directed mutagenesis to substitute each of these cysteines to alanine, thus generating five Cys-Ala surface mutants.

The extent of protein dimerisation in the wild-type P-450cam and the five surface Cys-Ala mutants were investigated, The presence of dimer was detected by both anion exchange fast protein liquid chromatography on a Resource Q column (Pharmacia) and gel filtration size exclusion chromatography on a Superose 12 column (Pharmacia) in the wild-type P-450cam and the C58A, C85A, C136A and C148A mutants. On the other hand, no dimer was detected, even at high concentrations (0.1 mM range), for the C334A mutant (see data in FIG. 2). We concluded that wild-type P-450cam underwent dimerisation by intermolecular S--S disulphide bond formation between the surface cysteine 334 on two protein molecules.

The C334A mutation has the obvious benefit of removing unwanted protein dimerisation, thus ensuring the presence of a single species in solution at all times. In addition, we noted a completely unexpected benefit of this mutation. Like all proteins, wild-type P-450cam shows aggregation upon standing. The reasons why proteins aggregate are not clear, but the P-450cam aggregates are insoluble and catalytically inactive. The wild-type and C58A, C85A, C136A and C148A mutants all showed dimerisation as well as aggregation upon storage at 4° C., and even in 50% glycerol solutions at -20° C. Aggregation will also occur during turnover, especially at the higher P-450cam concentrations required in any economically viable industrial application in, for example, synthesis of organic molecules. The C334A mutant did not show any evidence of aggregation even at mM concentrations at room temperature over a period of three days. Thus, the C334A mutation has beneficial effects in protein handling, storage, and increased catalyst lifetime.

We believe the mutation at position 96 to be the key which enables the mutant enzymes to catalyse the oxidation of a relatively wide range of organic substrates. Other amino acids adjacent to the active site of the enzyme may also be mutated in order to change the shape and specificity of the active site. These other amino acids include those at positions 87, 98, 101, 185, 193, 244, 247, 295, 297, 395 and 396. It is envisaged that the amino acid at one or more of these positions may be replaced by: a small hydrophobic amino acid so as to enlarge the active site; or a large hydrophobic amino acid so as to reduce the size of the active site; or by an amino acid having an aromatic ring to interact with a corresponding aromatic ring of a substrate.

Regarding the oxidation reactions, the conditions are described in the literature references attached. The enzyme system typically includes putidaredoxin and putidaredoxin reductase together with NADH as co-factors in addition to the mutant enzyme. The example of cyclohexylbenzene oxidation is described in the experimental section below. Various classes of organic compounds are envisaged and described below. We note that the wild-type P-450cam is active towards the oxidation of a number of molecules included in the following sections. However, in all cases the mutant P-450cam proteins show much higher turnover activities.

i) The organic compound is an aromatic compound, either a hydrocarbon or a compound used under conditions in which it does not inactivate or denature the enzyme. Since the mutation has been effected with a view to creating an aromatic-binding pocket in the active site of the enzyme, the mutant enzyme is capable of catalysing the oxidation of a wide variety of aromatic compounds. Oxidation of example aromatic and polyaromatic compounds is demonstrated in the experimental section below and is believed very surprising given that the wild-type enzyme has been reported to catalyse the oxidation of only members of the camphor family and shows low activity towards a few other molecules such as styrene¹⁹, ethylbenzene⁹.10, a tetralone derivative²⁰, and nicotine²¹.

ii) The organic compound may be a hydrocarbon, e.g. aliphatic or alicyclic, carrying a functional group (see Scheme 1). An aromatic protecting group is attached to the functional group prior to the oxidation reaction and removed from the functional group after the oxidation reaction. A suitable aromatic group is a benzyl group. The protecting group serves two purposes: firstly it makes the substrate more hydrophobic and hence increases binding to the hydrophobic enzyme pocket; secondly it may help to hold the substrate in place at the active site. Thus, with the correct aromatic protection group, both regio- and stereo-selective hydroxylation of the substrate may be achieved. Examples of monofunctionalised hydrocarbons are cyclohexyl, cyclopentyl and alkyl derivatives (Scheme 1). The oxidation products of these compounds are valuable starting materials for organic synthesis, particularly when produced in a homochiral form. A range of aromatic protecting groups are envisaged, e.g. benzyl or naphthyl ethers and benzoyl ethers and amides (Scheme 1). Of interest are also benzoxazole groups as carboxyl protecting groups and N-benzyl oxazolidine groups as aldehyde protecting groups. Both can be easily cleaved after the enzymatic oxidation and have previously been described in the literature for the microbial oxidations of aldehydes and acids²².

iii) The organic compound is a C4 to C12 aliphatic or alicyclic hydrocarbon. Oxidation of cyclohexane and linear and branched hydrocarbons is demonstrated in the experimental section below. We have found that wild-type P-450cam is also capable of oxidising these molecules, but the activities are low and in all cases the mutants show substantially higher activities.

iv) The organic compound is a halogenated aliphatic or alicyclic hydrocarbon. Oxidation of lindane (hexachlorocyclohexane) is also describe below.

Mutants were constructed in which active site substitutions were combined with the surface mutation of cysteine at position 334 to alanine and contained alanine, leucine, valine, or phenylalanine instead of tyrosine at position 96 (Y96). Lastly several active site mutations and the surface mutation were combined to constitute mutant enzymes with multiple mutations. The genes encoding cytochrome P-450cam, and its natural electron-transfer partners putidaredoxin and putidaredoxin reductase, were amplified from the total cellular DNA of P. Putida using the polymerise chain reaction (PCR). The expression vector/E. coli host combinations employed were pRH1091²³ in strain JM109 for P-450cam, pUC 118 in strain JM109 for putidaredoxin, and pGL W11 in strain DH5 for putidaredoxin reductase. Oligonucleotide-directed site-specific mutagenesis was carried out using an M13 mp 19 subclone by the method of Zoller and Smith²⁴, and mutant selection was by the method of Kunkel²⁵.

Binding of potential substrates was investigated by spectroscopic methods. The wild-type enzyme in the absence of substrate is in the 6-co-ordinated, low-spin form with a weakly bound water occupying the sixth co-ordination site, and shows a characteristic Soret maximum at 418 nm. Binding of camphor and the substrate analogues adamantanone, adamantane and norbornane fully converted the haem to the 5-co-ordinated, high-spin form which has a characteristic Soret band at 392 nm. This haem spin-state shift is accompanied by an increase in the haem reduction potential which enables the physiological electron-transfer partner putidaredoxin to reduce P-450cam and initiate the catalytic hydroxylation cycle²⁶. The haem spin state shift is thus a qualitative indication of the likelihood of molecules shown in Tables 1 and 2 being oxidised by the wild-type and mutant P-450cam enzymes.

A buffered solution (50 mM Tris.HCI, pH 7.4), typically 3 ml in volume, containing 10 uM putidaredoxin, 2 uM putidaredoxin reductase, 1 uM cytochrome P-450cam mono-oxygenase (wild-type or mutant), 200 mM KCI, 50 ug/ml bovine liver catalase (Sigma), and 1 mM target organic compound such as cyclohexylbenzene (added as a 0.1 M stock in ethanol) was preincubated at 30° C. for 5 minutes. The enzymatic reaction was initiated by adding NADH to a total concentration of 2 mM. Further four aliquots of NADH (to increase the NADH concentration by 1 mM each time) were added in intervals of 10 minutes, and 30 minutes into the incubation one aliquot of substrate (to increase the concentration by 1 mM) was also added. The reaction was quenched after 60 minutes by adding 0.5 ml chloroform and vortexing the mixture. The phases were separated by centrifugation (4000 g) at 4° C. The chloroform layer was analyzed by gas chromatography.

For many substrate compounds such as cyclohexylbenzene for which not all the P-450cam-mediated oxidation products are commercially available, the chloroform extracts are evaporated to dryness under a stream of nitrogen. The residues were extracted with hexane and the oxidation products separated by high performance liquid chromatography, eluting with a hexane/isopropanol gradient. The purified products were then identified by mass spectroscopy and particularly nuclear magnetic resonance spectroscopy.

For different substrates of different solubility in the aqueous buffer solution, the amount of substrate added to the incubation mixtures varies from 0.2 mM to 4 mM final concentration. The NADH concentration can be monitored at 340 nm and, in all cases, more substrates and NADH are added during the incubation.

Using the above experimental techniques, the inventors have investigated a considerable number of organic compounds as substrates for both the wild-type P-450cam enzyme and also the mutant version Y96A. Work has included mutants designated Y96V; Y96L; Y96f: C334A; the combined mutant F87A-Y96G-F193A and the combined active site and surface mutants of Y96A-C334A; Y96V-C334A; Y96L-C334A; Y96F-C334A; F87A-Y96G-F193A-C334A. The results for C334A and C334A-Y96A are set out in Table 1 and 2, in which structurally related molecules are grouped together.

Table 1 details the NADH consumption for oxidation of small linear, branched and cyclic hydrocarbons by the mutant Y96A-C334A. Tables 2(a) to 2(h) details the product distributions for mutant and substrate combinations where these have been elucidated to date.

The cysteine residue at position 344 can be deleted by any well known and freely available standard restriction techniques and will therefore not be described in detail herein.

    ______________________________________                                         Scheme 1:                                                                        Hydrocarbons                                                                   #STR1##                                                                         -                                                                             #STR2##                                                                         -                                                                           --Z     Protecting Group                                                       ______________________________________                                           --OH                                                                                                  #STR3##                                                                        #STR4##                                                  - --NH.sub.2                                                                                         #STR5##                                                  - --COOH                                                                                             #STR6##                                                                        #STR7##                                                  - --CHO                                                                                             ##STR8##                                               ______________________________________                                    

                  TABLE 1                                                          ______________________________________                                                             K.sub.app (μM).sup.a                                                        WT    Y96A                                                 ______________________________________                                                                             1 6.3 12                                      -                                                                                                               2 12 28                                       -                                                                                                               3 8.4 1.4                                     -                                                                                                               4 330 92                                      -                                                                                                               5 >1500.sup.b 73                           ______________________________________                                          .sup.a Values are the average of two independent measurements using the        method of Sligar (S. G. Sligar, Biochemistry, 1976, 15, 5399-5406). The        value of K.sub.app is strongly dependent on the concentration of K.sup.+       in the buffer. At [K.sup.+ ] > 150 mM, K.sub.app for camphor is 0.6 μM      for both wildtype and Y96A. Data in this table were determined at [K.sup.      ] = 70 mM in phosphate buffer, pH 7.4, in order to avoid  # salting out o      substrates at higher ion concentrations.                                       .sup.b Saturation not reached.                                           

                                      TABLE 2(a)                                   __________________________________________________________________________                         Wild type                                                                               Mutant Y96A                                                                             Wild type                                                                              Mutant Y96A                      P450cam-substrate interactions                                                                     ΔSpin                                                                             ΔSpin                                                                             NADH    NADH                               Subgroup: 1-ring high/low Vs DTT high/low Vs DTT turnover                                                                       ? GC? turnover?             __________________________________________________________________________                                                        GC?                                                                              Benzene -- -- -- --                                                            -                                                                              Toluene -- -- 30 30                                                            -                                                                              Ethylbenzene -- --                                                           40                             -                                                                                                                                Styrene -- -- 30 30                                                            -                                                                              Cyclohexene --  5 40                                                         40                             -                                                                                                                                1,3-Cyclohexadiene                                                           nd nd nd nd                    -                                                                                                                                1,4-Cyclohexadiene                                                           --  5 15 20                    -                                                                                                                                Cyclohexane -- -- 60                                                         60  +                          -                                                                                                                                Hexane -- -- 70 60                                                           +                              -                                                                                                                                Methylcyclohexane 50                                                         50 100  70                     -                                                                                                                                (S)-(+)-Carvone 10                                                           60 10 80                    __________________________________________________________________________

                                      TABLE 2(b)                                   __________________________________________________________________________                                 Wild type                                                                               Mutant Y96A                                                                              Wild type                                                                              Mutant Y96A             P450cam-substrate interactions                                                                             ΔSpin                                                                             ΔSpin                                                                              NADH    NADH                      Subgroup: 2-ring, Naphthalene high/low Vs DTT high/low Vs DTT turnover                                                                   ? GC?                                                                          turnover?          __________________________________________________________________________                                                                 GC?                                                                 Naphthalene -- -- 15 20                                                        -                                                                              1-Ethylnaphthalene -- --                                                      5 20                              -                                                                                                                            2-Ethylnaphthalene -- --                                                     10 20                              -                                                                                                                            2-Naphthylacetate -- 5                                                       --  5                              -                                                                                                                            1-Naphthylacetate -- 5                                                       --  5                              -                                                                                                                            1-Naphthylpropionate --                                                      20   0 20                          -                                                                                                                            1-Naphthylbutyrate -- 5                                                      --  5                              -                                                                                                                            Naphthylphenylketone --                                                      5 --  5                            -                                                                                                                            1,2-Dihydronaphthalene 5                                                     20  30 90                          -                                                                                                                            1,2,3,4-Tetrahydro                                                           naphthalene 5 10  40            __________________________________________________________________________                                                    40                         

                                      TABLE 2(c)                                   __________________________________________________________________________                                 Wild type                                                                               Mutant Y96A                                                                              Wild type                                                                              Mutant Y96A             P450cam-substrate interactions                                                                             ΔSpin                                                                             ΔSpin                                                                              NADH    NADH                      Subgroup: 2-ring, DPM high/low Vs DTT high/low Vs DTT turnover                                                                           ? GC?                                                                          turnover?          __________________________________________________________________________                                                                 GC?                                                                              Diphenylmeth                                                                 ane --  5 45                                                                   nd  + +                                                                          -                                                                              Diphenylethe                                                                 r 10  5 20 50         -                                                                                                                                         Benzophenone                                                                  -- 20 -- 20                                                                     -                                                                              Cyclohexylph                                                                 enylketone --                                                                  30 60 nd                                                                         -                                                                              Phenylbenzoa                                                                 te --  5 --                                                                    --                    -                                                                                                                                         N-Phenylbenz                                                                 ylamine   5                                                                    45 nd                                                                            -                                                                              Bibenzyl --                                                                  -- 55 55                                                                         -                                                                              cis-Stilbene                                                                  -- 20 40 50                                                                     -                                                                              Biphenyl --                                                                  20 -- 90                                                                         -                                                                              Cyclohexylbe                                                                 nzen 20 20 80                                                                  nd                    -                                                                                                                                         trans-Stilbe                                                                 ne -- -- --                                                                    --                    -                                                                                                                                         Benzylether                                                                  --  5 55           __________________________________________________________________________                                                                 nd            

                                      TABLE 2(d)                                   __________________________________________________________________________                                  Wild type                                                                               Mutant Y96A                                                                             Wild type                                                                              Mutant Y96A             P450cam-substrate interactions                                                                              ΔSpin                                                                             ΔSpin                                                                             NADH    NADH                      Subgroup: 3-ring high/low Vs DTT high/low Vs DTT turnover? GC? turnover?                                                                  GC?               __________________________________________________________________________                                                                   Anthracene                                                                     -                                                                              Phenanthrene                                                                  -- -- 20 20                                                                   +                     -                                                                                                                                         Fluorene --                                                                  -- -- 50                                                                         -                                                                              2-Fluorencar                                                                 boxaldehyde                                                                    -- -- -- 50                                                                      -                                                                              9-Fluorenone                                                                  -- 20 --  5                                                                     -                                                                              Anthrone --                                                                   5 --  5                                                                         -                                                                              Anthraquinon                                                                 e                     -                                                                                                                                         2-Ethylanthr                                                                 aquinone           __________________________________________________________________________

                                      TABLE 2(e)                                   __________________________________________________________________________                             Wild type                                                                               Mutant Y96A                                                                             Wild type                                                                              Mutant Y96A                  P450cam-substrate interactions                                                                         ΔSpin                                                                             ΔSpin                                                                             NADH    NADH                           Subgroup: 4,5-ring high/low Vs DTT high/low Vs DTT turnover                                                                         ? GC? turnover?         __________________________________________________________________________                                                            GC?                                                                 Chrsene -- -- -- --                   -                                                                                                                       1,2-Benzanthracene -- -- --                                                  --                                      -                                                                                                                       Fluoranthene -- 5 20 10                                                        -                                                                              Pyrene* -- -- -- --                   -                                                                                                                       Perylene* -- -- -- --              __________________________________________________________________________

                                      TABLE 2(f)                                   __________________________________________________________________________                                Wild type Mutant Y96A                                                                             Wild type                                                                               Mutant Y96A             P450cam-substrate interactions                                                                            ΔSpin                                                                              ΔSpin                                                                             NADH     NADH                      Subgroup: Cyclic Alkanes high/low Vs DTT high/low Vs DTT turnover                                                                        ? GC?                                                                          turnover?          __________________________________________________________________________                                                                 GC?                                                                              cis-Decahydr                                                                 onaphthalene                                                                   nd nd nd nd                                                                      -                                                                              trans-Decahy                                                                 dro  naphthale                                                                 ne 20 10 90                                                                    70                    -                                                                                                                                         Cyclohexane                                                                  -- -- 60 60                                                                    +                     -                                                                                                                                         Methylcycloh                                                                 exane 50 50                                                                    100  70            __________________________________________________________________________

                                      TABLE 2(g)                                   __________________________________________________________________________                                     Wild type                                                                      ΔSpin                                                                        Vs Mutant Y96A                                                                            Wild type                                                                              Mutant Y96A             P450cam-substrate interactions  high/                                                                              Vs ΔSpin                                                                         Vs NADH    NADH                      Subgroup: n-Alkanes low DTT high/low DTT turnover? GC? turnover?             __________________________________________________________________________                                                                 GC?                   n-Pentane -- 5 55 40  +                                                        n-Hexane -- -- 60 40  +                                                        n-Heptane 5 5 60 40  +                                                         n-Octane -- 5 80 45  +                                                         n-Nonane -- -- 70 45  +                                                        n-Decane nd nd nd nd                                                           n-Undecane nd nd 20 20                                                         n-Dodecane nd nd  5  5                                                        CH.sub.3 (CH.sub.2).sub.14 CH.sub.3 n-Hexadecane -- -- -- --                   CH.sub.3 (CH.sub.2).sub.15 CH.sub.3 n-Heptadecane -- -- -- --                  CH.sub.3 (CH.sub.2).sub.11 OSO.sub.3.Na SDS -- 20  -- 60                       CH.sub.3 (CH.sub.2).sub.7 CH═CH(CH.sub.2).sub.7 CO.sub.2 H Oleic                                                                     acid* -- 10?                                                                   --  20?                                                                         [(CH.sub.3).s                                                                 ub.2 CH(CH.sub                                                                 .2).sub.3                                                                      CH(CH.sub.3)(C                                                                 H.sub.2).sub.3                                                                  CH(CH.sub.3)C                                                                 H.sub.2                                                                        CH.sub.2                                                                       --].sub.2                                                                      Squalane --                                                                    -- -- 20                                                                         -                                                                              Isoprene --                                                                  -- 10              __________________________________________________________________________                                                                 10            

                                      TABLE 2(h)                                   __________________________________________________________________________                          Wild type                                                                               Mutant Y96A                                                                             Wild type                                                                              Mutant Y96A                     P450cam-substrate interactions                                                                      ΔSpin                                                                             ΔSpin                                                                             NADH    NADH                              Subgroup: Camphor-like high/low Vs DTT high/low Vs DTT turnover                                                                  ? GC? turnover?            __________________________________________________________________________                                                         GC?                                                                 (1R)-(##                                                                     -)-Camphorquinone 80 80 80 80                                                    -                                                                              (1R)-(-)-Fenchone 40 70 50 80                                                  -                                                                              Dicyclopentadiene 50 80 90            __________________________________________________________________________                                            90                                 

                  TABLE 3                                                          ______________________________________                                         Turnover of Small Alkanes by P450cam Mutants                                     All mutants listed below also contain the C334A mutation.                      Turnover rate measured as NADH consumption rate                                (nmole NADH/nmole P450cam/s).                                                  Alkane substrate:                                                            Main chain               Wild                                                    length Name type Y96A                                                        ______________________________________                                         C4       n-butane        --        --                                            C4 2-methyl butane background 4.6                                              C4 2,3-dimethyl butane background 16.8                                         C4 2,2-dimethyl butane background 14.0                                         C5 n-pentane background 5.8                                                    C5 2-methyl pentane 3.8 11.7                                                   C5 3-methyl pentane 1.3 14.2                                                   C5 2,4-dimethyl pentane 0.2 12.6                                               C5 2,2-dimethyl pentane 5.2 12.8                                               C5 2,2,4-trimethyl pentane 0.9 5.3                                             C5 3-ethyl pentane background 16.2                                             C6 n-hexane background 6.0                                                     C6 2-methyl hexane background 10.6                                             C7 n-heptane 2.7 4.4                                                           C7 2-methyl heptane background 2.1                                             C7 4-methyl heptane 1.4 10.2                                                   C8 n-octane background 5.8                                                     C7 cycloheptane 4.4 42.5                                                     ______________________________________                                          Product structures and distributions following oxidation of substrates         with P450cam active site mutants.                                              "background" - typical background NADH oxidation rate is 0.07 nmole NADH       (nmole P450cam).sup.-1 sec.sup.-1 -                                      

                                      TABLE 4(a)                                   __________________________________________________________________________     Product structure and distributions following oxidation of substrates          with P450cam                                                                     active site mutants. All mutants shown below also contain the C334A          mutation.                                                                      Cyclohexylbenzene    Products (%) for mutants:                                 Products             WT Y96A                                                                               Y96F                                                                              Y96L                                                                              Y96V                                         __________________________________________________________________________                                         3-ol 43 20 54 38 28                        L or D 3-ol 20 20 27 23 39                                                        -                                                                            ##STR69##        Trans-  4-ol 25 15  6 23 10                                     -                                                                                             ##S Cis-  4-ol 12 45 13 16 23                                   -                                                                           Total products(area/10.sup.5)                                                                       0.8                                                                               7.4 1.1                                                                               10.4                                                                              12.5                                           #STR71##                                                                        -                                                                             #STR72##                                                                        -                                                                            ##STR73##                                                                     __________________________________________________________________________

                  TABLE 4(b)                                                       ______________________________________                                                                 Products (%) for                                         Phenylcyclohexene mutants:                                                   Products                WT      Y96A                                           ______________________________________                                                                               3-one (A) 24 25                           ##STR75##          3-ol (B) 76 75                                                -                                                                           Total products(area/10.sup.6)                                                                          42      36                                               #STR76##                                                                        -                                                                               #STR77##                                                                      -                                                                                  ##STR78##                                                               ______________________________________                                    

                                      TABLE 4(c)                                   __________________________________________________________________________                    Products (%) for mutants:                                       Naphthalene                       F87A-F96G-                                     Products WT Y96A Y96F Y96L Y96V F193A                                        __________________________________________________________________________                                         1-ol 100 100 100 100 100 100                ##STR80##   2-ol 0 0 0 0 0 0                                                     -                                                                           Total products(area/10.sup.5)                                                                 (0.016)                                                                             1.1 2.4                                                                               0.7                                                                               1.4 0.1                                           ##STR81##                                                                     __________________________________________________________________________

                  TABLE 4(d)                                                       ______________________________________                                                Products (%) for mutants:                                               Phenanthrene                             F87A-F96G-                              Products WT Y96A Y96F Y96L Y96V F193A                                        ______________________________________                                         A        38      49      41   35.5 41    27                                      B 15 23 31 41 38 41                                                            C 12 13 5 9 11 3                                                               D 35 15 23 14.5 10 29                                                          Total products 0.075 7.0 4.5 2.8 1.6 0.065                                     (area/10.sup.6)                                                               ##STR82##                                                                     ______________________________________                                    

                  TABLE 4(e)                                                       ______________________________________                                                Products (%) for mutants:                                               Fluoranthene                             F87A-F96G-                              Products WT Y96A Y96F Y96L Y96V F193A                                        ______________________________________                                         A        0       84      --   --   --    0                                       B 0 16 -- -- -- 100                                                            Total products 0 2.7 -- -- -- 0.2                                              (area/10.sup.6)                                                               ##STR83##                                                                     ______________________________________                                    

                  TABLE 4(f)                                                       ______________________________________                                                Products (%) for mutants:                                               Pyrene                                   F87A-F96G-                              Products WT Y96A Y96F Y96L Y96V F193A                                        ______________________________________                                         A        0      40      43    23   30    33                                      B 0 43.6 29 64.5 55 40                                                         C 0 5 12.5 7.9 12 20                                                           D 0 11.4 15.5 4.6 3 7                                                          Total products 0 1.2 1.5 1.5 1.6 0.02                                          (area/10.sup.6)                                                               ##STR84##                                                                     ______________________________________                                    

                  TABLE 4(g)                                                       ______________________________________                                         Lindane Products   Products (%) for mutants                                    (hexachlorocyclohexane)                                                                           WT       Y96A                                               ______________________________________                                         A                  100      100                                                  Turnover rate 7.5 43.5                                                         nmole NADH (nmoleP450).sup.-1 s.sup.-1                                        ##STR85##                                                                     ______________________________________                                    

                  TABLE 4(h)                                                       ______________________________________                                                        Products(%) for mutants:                                                       Y96F   Y96A                                                     ______________________________________                                         Hexane Products                                                                  2-hexanone 10 15                                                               3-hexanone 16 28                                                               2-hexanol 24 26                                                                3-hexanol 50 32                                                                Relative activity 18.2 25.5                                                    (WT = 1)                                                                       2-Methyl hexane                                                                Products                                                                       2-methyl-2-hexanol 72 74                                                       5-methyl-2-hexanone 16 14                                                      2-methyl-3-hexanol 7 4                                                         5-methyl-2-hexanol 5 8                                                         Relative activity 2.3 2.6                                                      (WT = 1)                                                                     ______________________________________                                    

REFERENCES

1. "Cytochroe P-450: Structure, Mechanism, and Biochemistry", ed. P R Ortiz de Montellano, Plenum Press, New York,1986.

2. T L Poulos, B C Finzel and A J Howard, J. Mol. Biol., t987, 195, 687-700.

3. C A Hasemann, K G Ravichandran, J A Peterson, and J Deisenhofer, J. Mol. Biol., 1994, 2-)6, 1169-1185.

4. K G Ravichandran, S S Boddupalli, C A Hasemann, J A Peterson, and J Deisenhofer, Science, 1993, 261, 731-736.

5. B P Unger, I C Gunsalus, and S G Sligar, J. Biol. Chem., 1986, 261, 1158-1163; J S Miles, A W Munro, B N Rospendowski, W E Smith, J McKnight, and A J Thomson, Biochem. J., 1992, 288, 503-509; T H Richardson, M J Hsu, T Kronbach, H J Bames, G Chan, M R Waterman, B Kemper, and E F Johnson, Arch. Biochem. Biophys., 1993, 300, 510-516; S S Boddupalli, T Oster, R W Estabrook, and J A Peterson, J. Biol. Chem., 1992, 267, 10375-10380; H Li K Darish and T L Poulos. J. Biol. Chem., 1991, 266, 11909-11914.

6. I C Gunsalus and G C Wagner, Methods Enzymol., 1978, 52, 166-188.

7. M Haniu, L G Armes, K T Yasunobu, B A Shastry, and I C Gunsalus. Biol. Chem., 1982, 257, 12664-12671.

8. S G Sligar, D Filipovic, and P S Stayton, Methods Enzymol., 1991, 206, 31-49.

9. P J. Loida and S G Sligar, Biochemistrv, 1993, 32, 11530-11538.

10. P J Loida and S G Sligar, Protein Eng., 1993, 6, 207-212.

11. W M. Atkins and S G Sligar, J. Am. Chem. Soc., 1989, 111, 2715-2717.

12. S F Tuck, S Graham-Lorence, J A Peterson, and P R Ortiz de Montellano, J. Biol. Chem., 1993, 268, 269-275.

13. C Di Prime, G Hui Bin Hoa, P. Douzou, and S Sligar, J. Biol. Chem., 1990, 265, 5361-5363.

14. W M Atkins and S G Sligar, J. Biol. Chem., 1988, 263, 18842-18849.

15. W M Atkins and S G Sligar, Biochemistry, 1990, 29, 1271-1275.

16. J D Lipscomb, J E Harrison, K M Dus, and I C Gunsalus, Biochem. Biophys. Res. Conunun., 1978, 83, 771-778.

17. P J Goodford, J. Med. Chem., 1985, 28, 849-857.

18. Quanta 4.0, Molecular Simulations Inc., 16 New England Executive Park, Burlington, Mass. 01803-5297.

19. J A Fruetet, J R Collins, D L Camper, G H Loew, and P R Ortiz de Montallano, J. Am. Chem. Soc., 1992, 114, 6987-6993.

20. Y Watanabe and Y Ishimura, J. Am. Chem. Sec., 1989, 111, 410-41 1.

21. J P Jones, W F Trager, and T J Carlson, J. Am. Chem. Soc., 1993, 115, 381-387.

22. "Biotransformation in Preparative Organic Chemistry" H G Davis, R H Green, D R Kelly, and S M Roberts, Academic Press, London, 1989, Page 169 ff.

23. J E Baldwin J M Blackburn, R J Heath, and J D Sutherland, Bioorg. Med. Chem. Letts. 1992, 2, 663-668.

24. M J Zoller and M Smith, Nucleic Acids Res., 1982, 10, 6487-6500.

25. T A Kunkel, Proc. Natl. Acad. Sci., USA 1985, 82, 488-492.

26. S G Sligar and I C Gunsalus, Proc. Natl. Acad. Sci., USA, 1976, 73, 1078-1082.

    __________________________________________________________________________     #             SEQUENCE LISTING                                                    - -  - - <160> NUMBER OF SEQ ID NOS: 2                                         - - <210> SEQ ID NO 1                                                         <211> LENGTH: 1242                                                             <212> TYPE: DNA                                                                <213> ORGANISM: Pseudomonas putida                                             <220> FEATURE:                                                                 <221> NAME/KEY: P450cam WT                                                     <222> LOCATION: 1...1242                                                        - - <400> SEQUENCE: 1                                                          - - acg act gaa acc ata caa agc aac gcc aat ct - #t gcc cct ctg cca         ccc       48                                                                     Thr Thr Glu Thr Ile Gln Ser Asn Ala Asn Le - #u Ala Pro Leu Pro Pro           1               5   - #               10   - #                15                - - cat gtg cca gag cac ctg gta ttc gac ttc ga - #c atg tac aat ccg tcg            96                                                                        His Val Pro Glu His Leu Val Phe Asp Phe As - #p Met Tyr Asn Pro Ser                        20      - #            25      - #            30                    - - aat ctg tct gcc ggc gtg cag gag gcc tgg gc - #a gtt ctg caa gaa tca           144                                                                        Asn Leu Ser Ala Gly Val Gln Glu Ala Trp Al - #a Val Leu Gln Glu Ser                    35          - #        40          - #        45                        - - aac gta ccg gat ctg gtg tgg act cgc tgc aa - #c ggc gga cac tgg atc           192                                                                        Asn Val Pro Asp Leu Val Trp Thr Arg Cys As - #n Gly Gly His Trp Ile                50              - #    55              - #    60                            - - gcc act cgc ggc caa ctg atc cgt gag gcc ta - #t gaa gat tac cgc cac           240                                                                        Ala Thr Arg Gly Gln Leu Ile Arg Glu Ala Ty - #r Glu Asp Tyr Arg His            65                  - #70                  - #75                  - #80         - - ttt tcc agc gag tgc ccg ttc atc cct cgt ga - #a gcc ggc gaa gcc tac           288                                                                        Phe Ser Ser Glu Cys Pro Phe Ile Pro Arg Gl - #u Ala Gly Glu Ala Tyr                            85  - #                90  - #                95                - - gac ttc att ccc acc tcg atg gat ccg ccc ga - #g cag cgc cag ttt cgt           336                                                                        Asp Phe Ile Pro Thr Ser Met Asp Pro Pro Gl - #u Gln Arg Gln Phe Arg                        100      - #           105      - #           110                   - - gcg ctg gcc aac caa gtg gtt ggc atg ccg gt - #g gtg gat aag ctg gag           384                                                                        Ala Leu Ala Asn Gln Val Val Gly Met Pro Va - #l Val Asp Lys Leu Glu                    115          - #       120          - #       125                       - - aac cgg atc cag gag ctg gcc tgc tcg ctg at - #c gag agc ctg cgc ccg           432                                                                        Asn Arg Ile Gln Glu Leu Ala Cys Ser Leu Il - #e Glu Ser Leu Arg Pro                130              - #   135              - #   140                           - - caa gga cag tgc aac ttc acc gag gac tac gc - #c gaa ccc ttc ccg ata           480                                                                        Gln Gly Gln Cys Asn Phe Thr Glu Asp Tyr Al - #a Glu Pro Phe Pro Ile            145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - cgc atc ttc atg ctg ctc gca ggt cta ccg ga - #a gaa gat atc ccg         cac      528                                                                     Arg Ile Phe Met Leu Leu Ala Gly Leu Pro Gl - #u Glu Asp Ile Pro His                           165  - #               170  - #               175               - - ttg aaa tac cta acg gat cag atg acc cgt cc - #g gat ggc agc atg acc           576                                                                        Leu Lys Tyr Leu Thr Asp Gln Met Thr Arg Pr - #o Asp Gly Ser Met Thr                        180      - #           185      - #           190                   - - ttc gca gag gcc aag gag gcg ctc tac gac ta - #t ctg ata ccg atc atc           624                                                                        Phe Ala Glu Ala Lys Glu Ala Leu Tyr Asp Ty - #r Leu Ile Pro Ile Ile                    195          - #       200          - #       205                       - - gag caa cgc agg cag aag ccg gga acc gac gc - #t atc agc atc gtt gcc           672                                                                        Glu Gln Arg Arg Gln Lys Pro Gly Thr Asp Al - #a Ile Ser Ile Val Ala                210              - #   215              - #   220                           - - aac ggc cag gtc aat ggg cga ccg atc acc ag - #t gac gaa gcc aag agg           720                                                                        Asn Gly Gln Val Asn Gly Arg Pro Ile Thr Se - #r Asp Glu Ala Lys Arg            225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - atg tgt ggc ctg tta ctg gtc ggc ggc ctg ga - #t acg gtg gtc aat         ttc      768                                                                     Met Cys Gly Leu Leu Leu Val Gly Gly Leu As - #p Thr Val Val Asn Phe                           245  - #               250  - #               255               - - ctc agc ttc agc atg gag ttc ctg gcc aaa ag - #c ccg gag cat cgc cag           816                                                                        Leu Ser Phe Ser Met Glu Phe Leu Ala Lys Se - #r Pro Glu His Arg Gln                        260      - #           265      - #           270                   - - gag ctg atc gag cgt ccc gag cgt att cca gc - #c gct tgc gag gaa cta           864                                                                        Glu Leu Ile Glu Arg Pro Glu Arg Ile Pro Al - #a Ala Cys Glu Glu Leu                    275          - #       280          - #       285                       - - ctc cgg cgc ttc tcg ctg gtt gcc gat ggc cg - #c atc ctc acc tcc gat           912                                                                        Leu Arg Arg Phe Ser Leu Val Ala Asp Gly Ar - #g Ile Leu Thr Ser Asp                290              - #   295              - #   300                           - - tac gag ttt cat ggc gtg caa ctg aag aaa gg - #t gac cag atc ctg cta           960                                                                        Tyr Glu Phe His Gly Val Gln Leu Lys Lys Gl - #y Asp Gln Ile Leu Leu            305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - ccg cag atg ctg tct ggc ctg gat gag cgc ga - #a aac gcc tgc ccg         atg     1008                                                                     Pro Gln Met Leu Ser Gly Leu Asp Glu Arg Gl - #u Asn Ala Cys Pro Met                           325  - #               330  - #               335               - - cac gtc gac ttc agt cgc caa aag gtt tca ca - #c acc acc ttt ggc cac          1056                                                                        His Val Asp Phe Ser Arg Glu Lys Val Ser Hi - #s Thr Thr Phe Gly His                        340      - #           345      - #           350                   - - ggc agc cat ctg tgc ctt ggc cag cac ctg gc - #c cgc cgg gaa atc atc          1104                                                                        Gly Ser His Leu Cys Leu Gly Gln His Leu Al - #a Arg Arg Glu Ile Ile                    355          - #       360          - #       365                       - - gtc acc ctc aag gaa tgg ctg acc agg att cc - #t gac ttc tcc att gcc          1152                                                                        Val Thr Leu Lys Glu Trp Leu Thr Arg Ile Pr - #o Asp Phe Ser Ile Ala                370              - #   375              - #   380                           - - ccg ggt gcc cag att cag cac aag agc ggc at - #c gtc agc ggc gtg cag          1200                                                                        Pro Gly Ala Gln Ile Gln His Lys Ser Gly Il - #e Val Ser Gly Val Gln            385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - gca ctc cct ctg gtc tgg gat ccg gcg act ac - #c aaa gcg gta                  - #1242                                                                    Ala Leu Pro Leu Val Trp Asp Pro Ala Thr Th - #r Lys Ala Val                                    405  - #               410  - #           414                   - -  - - <210> SEQ ID NO 2                                                    <211> LENGTH: 1242                                                             <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <221> NAME/KEY: P450cam C334A                                                  <222> LOCATION: 1 1242                                                         <223> OTHER INFORMATION: Mutant with Cys-334 (tgc - #at nucleotides 1000             1002) substituted with Ala                                                - - <400> SEQUENCE: 2                                                          - - acg act gaa acc ata caa agc aac gcc aat ct - #t gcc cct ctg cca ccc            48                                                                        Thr Thr Glu Thr Ile Gln Ser Asn Ala Asn Le - #u Ala Pro Leu Pro Pro            1               5   - #               10   - #                15                - - cat gtg cca gag cac ctg gta ttc gac ttc ga - #c atg tac aat ccg tcg            96                                                                        His Val Pro Glu His Leu Val Phe Asp Phe As - #p Met Tyr Asn Pro Ser                        20      - #            25      - #            30                    - - aat ctg tct gcc ggc gtg cag gag gcc tgg gc - #a gtt ctg caa gaa tca           144                                                                        Asn Leu Ser Ala Gly Val Gln Glu Ala Trp Al - #a Val Leu Gln Glu Ser                    35          - #        40          - #        45                        - - aac gta ccg gat ctg gtg tgg act cgc tgc aa - #c ggc gga cac tgg atc           192                                                                        Asn Val Pro Asp Leu Val Trp Thr Arg Cys As - #n Gly Gly His Trp Ile                50              - #    55              - #    60                            - - gcc act cgc ggc caa ctg atc cgt gag gcc ta - #t gaa gat tac cgc cac           240                                                                        Ala Thr Arg Gly Gln Leu Ile Arg Glu Ala Ty - #r Glu Asp Tyr Arg His            65                  - #70                  - #75                  - #80         - - ttt tcc agc gag tgc ccg ttc atc cct cgt ga - #a gcc ggc gaa gcc tac           288                                                                        Phe Ser Ser Glu Cys Pro Phe Ile Pro Arg Gl - #u Ala Gly Glu Ala Tyr                            85  - #                90  - #                95                - - gac ttc att ccc acc tcg atg gat ccg ccc ga - #g cag cgc cag ttt cgt           336                                                                        Asp Phe Ile Pro Thr Ser Met Asp Pro Pro Gl - #u Gln Arg Gln Phe Arg                        100      - #           105      - #           110                   - - gcg ctg gcc aac caa gtg gtt ggc atg ccg gt - #g gtg gat aag ctg gag           384                                                                        Ala Leu Ala Asn Gln Val Val Gly Met Pro Va - #l Val Asp Lys Leu Glu                    115          - #       120          - #       125                       - - aac cgg atc cag gag ctg gcc tgc tcg ctg at - #c gag agc ctg cgc ccg           432                                                                        Asn Arg Ile Gln Glu Leu Ala Cys Ser Leu Il - #e Glu Ser Leu Arg Pro                130              - #   135              - #   140                           - - caa gga cag tgc aac ttc acc gag gac tac gc - #c gaa ccc ttc ccg ata           480                                                                        Gln Gly Gln Cys Asn Phe Thr Glu Asp Tyr Al - #a Glu Pro Phe Pro Ile            145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - cgc atc ttc atg ctg ctc gca ggt cta ccg ga - #a gaa gat atc ccg         cac      528                                                                     Arg Ile Phe Met Leu Leu Ala Gly Leu Pro Gl - #u Glu Asp Ile Pro His                           165  - #               170  - #               175               - - ttg aaa tac cta acg gat cag atg acc cgt cc - #g gat ggc agc atg acc           576                                                                        Leu Lys Tyr Leu Thr Asp Gln Met Thr Arg Pr - #o Asp Gly Ser Met Thr                        180      - #           185      - #           190                   - - ttc gca gag gcc aag gag gcg ctc tac gac ta - #t ctg ata ccg atc atc           624                                                                        Phe Ala Glu Ala Lys Glu Ala Leu Tyr Asp Ty - #r Leu Ile Pro Ile Ile                    195          - #       200          - #       205                       - - gag caa cgc agg cag aag ccg gga acc gac gc - #t atc agc atc gtt gcc           672                                                                        Glu Gln Arg Arg Gln Lys Pro Gly Thr Asp Al - #a Ile Ser Ile Val Ala                210              - #   215              - #   220                           - - aac ggc cag gtc aat ggg cga ccg atc acc ag - #t gac gaa gcc aag agg           720                                                                        Asn Gly Gln Val Asn Gly Arg Pro Ile Thr Se - #r Asp Glu Ala Lys Arg            225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - atg tgt ggc ctg tta ctg gtc ggc ggc ctg ga - #t acg gtg gtc aat         ttc      768                                                                     Met Cys Gly Leu Leu Leu Val Gly Gly Leu As - #p Thr Val Val Asn Phe                           245  - #               250  - #               255               - - ctc agc ttc agc atg gag ttc ctg gcc aaa ag - #c ccg gag cat cgc cag           816                                                                        Leu Ser Phe Ser Met Glu Phe Leu Ala Lys Se - #r Pro Glu His Arg Gln                        260      - #           265      - #           270                   - - gag ctg atc gag cgt ccc gag cgt att cca gc - #c gct tgc gag gaa cta           864                                                                        Glu Leu Ile Glu Arg Pro Glu Arg Ile Pro Al - #a Ala Cys Glu Glu Leu                    275          - #       280          - #       285                       - - ctc cgg cgc ttc tcg ctg gtt gcc gat ggc cg - #c atc ctc acc tcc gat           912                                                                        Leu Arg Arg Phe Ser Leu Val Ala Asp Gly Ar - #g Ile Leu Thr Ser Asp                290              - #   295              - #   300                           - - tac gag ttt cat ggc gtg caa ctg aag aaa gg - #t gac cag atc ctg cta           960                                                                        Tyr Glu Phe His Gly Val Gln Leu Lys Lys Gl - #y Asp Gln Ile Leu Leu            305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - ccg cag atg ctg tct ggc ctg gat gag cgc ga - #a aac gcc gcc ccg         atg     1008                                                                     Pro Gln Met Leu Ser Gly Leu Asp Glu Arg Gl - #u Asn Ala Ala Pro Met                           325  - #               330  - #               335               - - cac gtc gac ttc agt cgc caa aag gtt tca ca - #c acc acc ttt ggc cac          1056                                                                        His Val Asp Phe Ser Arg Glu Lys Val Ser Hi - #s Thr Thr Phe Gly His                        340      - #           345      - #           350                   - - ggc agc cat ctg tgc ctt ggc cag cac ctg gc - #c cgc cgg gaa atc atc          1104                                                                        Gly Ser His Leu Cys Leu Gly Gln His Leu Al - #a Arg Arg Glu Ile Ile                    355          - #       360          - #       365                       - - gtc acc ctc aag gaa tgg ctg acc agg att cc - #t gac ttc tcc att gcc          1152                                                                        Val Thr Leu Lys Glu Trp Leu Thr Arg Ile Pr - #o Asp Phe Ser Ile Ala                370              - #   375              - #   380                           - - ccg ggt gcc cag att cag cac aag agc ggc at - #c gtc agc ggc gtg cag          1200                                                                        Pro Gly Ala Gln Ile Gln His Lys Ser Gly Il - #e Val Ser Gly Val Gln            385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - gca ctc cct ctg gtc tgg gat ccg gcg act ac - #c aaa gcg gta                  - #1242                                                                    Ala Leu Pro Leu Val Trp Asp Pro Ala Thr Th - #r Lys Ala Val                                    405  - #               410  - #           414                 __________________________________________________________________________ 

What is claimed is:
 1. A mutant mono-oxygenase cytochrome P450cam comprising either a deletion of the cysteine at amino acid position 334 or a substitution of another amino acid for the cysteine at amino acid position
 334. 2. A mutant mono-oxygenase cytochrome P450cam according to claim 1 wherein an amino acid other than cysteine is substituted at amino acid position
 334. 3. A mutant mono-oxygenase cytochrome P450cam according to claim 2 further comprising the substitution of an amino acid other than tyrosine at amino acid position
 96. 4. A mutant mono-oxygenase cytochrome P⁴⁵⁰ cam according to claim 2 further comprising one or more amino acid substitutions at amino acid positions selected from the group consisting of 87, 98, 101, 185, 193, 244, 247, 295, 297, 395, and
 396. 5. A mutant mono-oxygenase cytochrome P450cam according to claim 1 wherein the cysteine is deleted at amino acid position
 334. 6. A mutant mono-oxygenase cytochrome P450cam according to claim 5 further comprising the substitution of an amino acid other than tyrosine at amino acid position
 96. 7. A mutant mono-oxygenase cytochrome P450cam according to claim 5 further comprising one or more amino acid substitutions at amino acid positions selected from the group consisting of 87, 98, 101, 185, 193, 244., 247, 295, 297, 395, and
 396. 8. A mutant mono-oxygenase cytochrome P450cam according to claim 1 further comprising the substitution of an amino acid other than tyrosine at amino acid position
 96. 9. A mutant mono-oxygenase cytochrome P450cam according to claim 8 wherein the substituent amino acid for position 96 is selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine and valine.
 10. A mutant mono-oxygenase cytochrome P450cam according to claim 8 further comprising one or more amino acid substitutions at amino acid positions selected from the group consisting of 87, 98, 101, 185, 193, 244, 247, 295, 297, 395, and
 396. 11. A mutant mono-oxgenase cytochrome P450cam according to claim 1 wherein the substituent amino acid is selected from the group consisting of alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, histidine, isoleucilne, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine and valine.
 12. A mutant mono-oxygenase cytochrome P450cam according to claim 11 further comprising one or more amino acid substitutions at amino acid positions selected from the group consisting of 87, 98, 101, 185, 193, 244, 247, 295, 297, 395, and
 396. 13. A mutant mono-oxygenase cytochrome P450cam according to claim 1 further comprising one or more amino acid substitutions at amino acid positions selected from the group consisting of 87, 98, 101, 185, 193, 244, 247, 295, 297, 395, and
 396. 